Cellular interactions which occur during an immune response are regulated by members of several families of cell surface receptors, including the tumor necrosis factor receptor (TNFR) family. The TNFR family consists of a number of integral membrane glycoprotein receptors many of which, in conjunction with their respective ligands, regulate interactions between different hematopoietic cell lineages (Smith et al., The TNF Receptor Superfamily of Cellular and Viral Proteins: Activation, Costimulation and Death, 76:959–62, 1994; Cosman, Stem Cells 12:440–55, 1994).
The TNF receptor family is composed of a number of type I integral membrane glycoproteins which exhibit sequence homology, particularly with respect to cysteine-rich repeats in their extracellular domains. The TNF receptor family includes p75 NGFR (Johnson et al., Cell 47:545–54, 1989), p55 TNFR-I (Loetscher et al., Cell 61:351–59, 1990), p75 TNFR-II (Schall et al., Cell 61:361–70, 1990), TNFR-RP/TNFR-III (Crowe et al. Science 264:707–10, 1994), CD27 (Camerini et al., J. Immunol. 147:3165–69, 1991), CD30 (Falini et al., Blood 85:1–14, 1995), CD40 (Clark and Lane, Annu. Rev. Immunol. 9:97–127, 1991), 4-1BB (Kwon and Weissman, Proc. Natl. Acad. Sci. USA 86:1963–67, 1989; Schwarz et al., Gene 134:295–298, 1993), OX40 (Malletet al., EMBO J. 9:1063–68, 1990), FAS/APO-1 (Itoh et al., Cell 66:233–43, 1991), DR3 (Chinnaiyan et al., Science 274:990–92, 1996) also known as WSL-1 (Kitson et al., Nature 384:327–75, 1996), DR4 (Pan et al., Science 276:111–13, 1997), DR5 (Pan et al., Science 277:815–8, 1997; Sheridan et al., Science 277:818–21, 1997), osteoprotegerin (OPG) (Simonet et al., Cell 89:309–19, 1997; Human Genome Science, WIPO Publication WO96/28546), CAR1, found in chickens (Brojatsch et al., Cell 87:845–55, 1996), TRID or DcR1 (Pan et al., Science 277:815–8, 1997; Sheridan et al., Science 277:818–21, 1997) plus several viral open reading frames encoding TNFR-related molecules. NGFR, TNFR-I, CD30, CD40, 4-1BB, DR3 and OX40 are mainly restricted to cells of the lymphoid/hematopoietic system. TNFR-I, TNFR-II, TNFR-III and DR4 are found in most human tissues.
Members of the TNF receptor family are characterized by a multi-domain structure comprising an extracellular region, a transmembrane domain, a linker region between the extracellular ligand-binding region and the transmembrane domain and a cytoplasmic domain, which in several members of this family (TNFR 1, Fas, DR3, DR4, DR5, CAR1 and low affinity NGFR) contains a death domain associated with apoptosis. One member, TRID or DcR1 (Pan et al., Science 277:815–8, 1997; Sheridan et al., ibid.) has a hydrophobic N-terminus with cysteine rich repeats #2 and #3 followed by five tandem repeats of 15 amino acid residues which concludes with a transmembrane domain. The extracellular ligand-binding region is characterized by the presence of one to six cysteine-rich motifs each containing about six cysteines and approximately 40 amino acids, although variation in the size and number of these motifs occurs among members of this family. The cysteine-rich regions provide the motif for binding to shared structures in the ligands. The highest degree of homology among the TNFR family members is within this extracellular cysteine-rich region. Among human TNFRs the average homology is in the range of 25% to 30%. Between the last cysteine-rich repeat and the transmembrane domain is a small spacer region of between 8 to 70 amino acid residues. Cell surface TNF receptors are anchored in the cell membrane by a transmembrane domain characterized by a sequence of hydrophobic amino acid residues. On the opposite end of the protein from the extracellular ligand-binding region and separated from it by the transmembrane domain is the cytoplasmic domain. The cytoplasmic domains of TNFR family members are small, from 46 to 221 amino acid residues, which suggests possible differences in the signaling mechanisms among family members. In the TNF receptor for example, activation is triggered by the aggregation of cytoplasmic domains of three receptors when their corresponding extracellular domains bind to trimeric ligand.
One member of the TNF receptor family, osteoprotegerin (Simonet et al., ibid.), is unique in that it is a secreted protein. Soluble forms of other TNF receptors have been described for TNFR-I, TNFR-II, low-affinity NGFR, FAS, CD27, CD30, CD40 and 4-1BB, but these were generated either by cleaving from the cell membrane or secreted by alternatively spliced mRNA. OPG inhibits osteoclast maturation and it is thought that it might serve to regulate bone density by modulating osteoclast differentiation from hematopoietic precursors. OPG provided protection from normal osteoclast remodeling and ovariectomy-associated bone loss.
Ligands for these receptors have been identified, and with one exception (NGF) belong to the TNF ligand family. The members of the TNF ligand family share approximately 20% sequence identity in the extracellular ligand-binding regions, and exist mainly as type II membrane glycoproteins, biologically active as trimeric or multimeric complexes. This group includes TNF, LT-a, LT-b (Browning et al., Cell 72:847–56, 1993), CD27L (Goodwin et al., Cell 73:447–56, 1993), CD30L (Smith et al., Cell 73:1349–60, 1993), CD40L (Armitage et al., Nature 357:80–82, 1992), 4-1BBL (Goodwin et al., Eur. J, Immunol. 23:2631–41, 1993), OX40L (Godfrey et al., J. Exp. Med. 180:757–62, 1994), TRAIL or apo-2 (Wiley et al., Immunity 3:673–82, 1995), TNFg (Human Genome Sciences, WIPO Publication WO96/14328) and FasL (Cosman, ibid., Lotz et al., J. Leuko. Biol. 60:1–7, 1996). Soluble ligand forms have been identified for TNF, LT-a and FasL. It is not known whether a specific protease cleaves each ligand, releasing it from the membrane, or whether one protease serves the same function for all TNF ligand family members. TACE (TNF-alpha converting enzyme) has been shown to cleave TNF (Moss et al., Nature 385:733–36, 1997; Black et al., Nature 385:729–33, 1997). No other such enzymes are known.
The X-ray crystallographic structures have been resolved for human TNF (Jones et al., Nature 338:225–28, 1989), LT-a (Eck et al., J. Biol. Chem. 267:2119–122, 1992) and the LT-a/TNFR complex (Banner et al., Cell 73:431–45, 1993). This complex features three receptor molecules bound symmetrically to one LT-a trimer. A model of trimeric ligand binding through receptor oligomerization has been proposed to initiate signal transduction pathways. The identification of biological activity of several TNF members has been facilitated through use of monoclonal antibodies specific for the corresponding receptor. These monoclonal antibodies tend to be stimulatory when immobilized and antagonistic in soluble form. This is further evidence that receptor crosslinking is a prerequisite for signal transduction in this receptor family. Importantly, the use of receptor-specific monoclonal antibodies or soluble receptors in the form of multimeric Ig fusion proteins has been useful in determining biological function in vitro and in vivo for several family members. Soluble receptor-Ig fusion proteins have been used successfully in the cloning of the cell surface ligands corresponding to the CD40, CD30, CD27, 4-1BB and Fas receptors.
In general, the members of the tumor necrosis factor ligand family mediate interactions between different hematopoietic cells, such as T cell/B cell, T cell/monocyte and T cell/T cell interactions. The result of this two-way communication can be stimulatory or inhibitory, depending on the target cell or the activation state. These TNF proteins are involved in regulation of cell proliferation, activation and differentiation, including control of cell survival or death by apoptosis or cytotoxicity. One member of this family, OX-40, is restricted to T cells where it acts as a costimulatory receptor. However, among the TNFR family members there are differences in distribution, kinetics of induction and requirements for induction, which support a defined role for each of the ligands in T cell-mediated immune responses.
The demonstrated in vitro and in vivo activities of these TNF receptor ligand family members illustrate the enormous clinical potential of, and need for, other TNF receptors, TNF ligands, TNFR agonists, and TNFR antagonists. The present invention addresses this need by providing a novel TNF receptor and related compositions and methods.